Two-dimensional gas-phase separations based on ion mobility (IM)-time-of-flight mass spectrometry (TOFMS) have demonstrated unique potential in the analysis of a wide range of materials and more recently in the analysis of complex mixtures of biomolecules [T. Wyttenbach and M. T. Bowers, Gas-Phase Conformations: The Ion Mobility/Ion Chromatography Method, Top. Curr. Chem. 225, 207-232 (2003) and references therein; and C. S. Hoaglund-Hyzer, A. E. Counterman, and D. E. Clemmer, Anhydrous Protein Ions, Chem. Rev. 99, 3037-3079 (1999) and references therein.]
Gas-phase ion mobility (IM) provides ion separation by generating or injecting ions (and gaseous neutral species) in/into a gas-filled drift tube (typically 1 to 760 Torr) where they migrate under the influence of a weak electrostatic-field (typically 1 to 100 V cm−1 Torr-1) and are impeded by collisions with the background gas. Biologically relevant ions are injected into the drift cell by using pulsed ion sources (e.g., matrix assisted laser desorption/ionization (MALDI)) or by pulsing a continuous ion source (e.g., electrospray (ESI) or ion spray). Other techniques to generate biologically relevant ions (and gaseous neutral species) may be used, such as surface enhanced laser desorption/ionization (SELDI). Other nonlimiting examples include atmospheric pressure MALDI, ultraviolet MALDI, infrared MALDI, direct LDI (laser desorption/ionization), nanospray, photoionization, multiphoton ionization, resonance ionization, thermal ionization, surface ionization, electric field ionization, chemical ionization, atmospheric pressure chemical ionization, radioactive ionization, discharge arc/spark ionization, laser induced breakdown ionization, inductively coupled plasma ionization, direct current plasma ionization, capacitively coupled plasma ionization, glow discharge ionization, microwave plasma ionization, and any combinations thereof. The theory of IM is fully developed in texts by Mason and McDaniel [E. W. McDaniel and E. A. Mason, The Mobility and Diffusion of Ions in Gases, Wiley, New York, N.Y. (1973); E. A. Mason and E. W. McDaniel, Transport Properties of Ions in Gases, John Wiley & Sons, Inc., New York, N.Y. (1988)], and the combination of IM with quadrupole mass spectrometry and subsequently time-of-flight mass spectrometry (TOFMS) dates back to the early 1960's [W. S. Barnes, D. W. Martin, and E. W. McDaniel, Mass Spectrographic Identification of the Ion Observed in Hydrogen Mobility Experiments, Phys. Rev. Lett. 6, 110-111 (1961); K. B. McAfee Jr. and D. Edelson, Identification and Mobility of Ions in a Townsend Discharge by Time-Resolved Mass Spectrometry, Proc. Phys. Soc. London 81, 382-384 (1963)]. The mobility (K) of an ion is determined by the ratio of the drift velocity (vd) to the electric field strength (E):
                    K        =                              v            d                    E                                    [        1        ]            
When the ion-neutral collision energy nears the thermal energy of the system, the mobility approaches the so-called “low-field” limit and can be related to the collision cross-section (Ω), or apparent surface area, of the ion:
                    K        =                              3            16                    ⁢                      q            N                    ⁢                                    (                                                1                  μ                                ⁢                                                      2                    ⁢                    π                                                                              k                      B                                        ⁢                    T                                                              )                                      1              2                                ⁢                      1            Ω                                              [        2        ]            
Where N is the number density of the drift gas, q is the ion charge (in MS techniques this is typically termed ze), μ is the reduced mass of the ion-neutral collision pair, kb is Boltzmann's constant, and T is the temperature of the system. Thus, IM provides separation selectivity based on the charge-to-collision cross-section (q/Ω) ratio of the analyte ion in a particular background drift gas, in contrast with MS based ion separation, which separates analyte ions on the basis of their mass-to-charge (m/z) ratio.
Analyte selectivity based on ion mobility separation provides several important advantages over prior art solution-based purification (e.g., high performance liquid chromatography) or gas-based mass-to-charge selection (i.e., MS) of biological molecules: (i) in many cases isobaric and isoform species (e.g., structural and/or conformational isomers) can be separated [F. W. Karasek and D. M. Kane, Plasma Chromatography of Isomeric Halogenated Nitrobenzenes, Anal. Chem. 46, 780-782 (1974); J. C. Tou and G. U. Boggs, Determination of Sub Parts-Per-Million Levels of Sec-butyl Chloropiphenyl Oxides in Biological Tissues by Plasma Chromatography, Anal. Chem. 48, 1351-1357 (1976); T. W. Carr, Plasma Chromatography of Isomeric Dihalogenated Benzene, J. Chrom. Sci. 15, 85-88 (1977); D. F. Hagen, Characterization of Isomeric Compounds by Gas and Plasma Chromatography, Anal. Chem. 51, 870-874 (1979)], (ii) the separation mechanism does not rely on solution-phase physical properties (e.g., hydropathy, isoelectric point, affinity, etc.) [E. W. McDaniel and E. A. Mason, The Mobility and Diffusion of Ions in Gases, Wiley, New York, N.Y. (1973); E. A. Mason and E. W. McDaniel, Transport Properties of Ions in Gases, John Wiley & Sons, Inc., New York, N.Y. (1988)], (iii) it is amenable to a wide variety of molecular classes or complex mixtures thereof (e.g., proteins, lipids, oligonucleotides, carbohydrates, etc.) [J. M. Koomen, B. T. Ruotolo, K. J. Gillig, J. A. McLean, D. H. Russell, M. Kang, K. R. Dunbar, K. Fuhrer, M. Gonin, and J. A. Schultz, Oligonucleotide Analysis with MALDI-Ion Mobility-TOFMS, Anal. Bioanal. Chem. 373, 612-617 (2002)], and (iv) in many cases it is sensitive and selective for post-translationally modified peptides (or proteins) [B. T. Ruotolo, G. F. Verbeck, L. M. Thompson, A. S. Woods, K. J. Gillig, and D. H. Russell, Distinguishing Between Phosphorylated and Nonphosphorylated Peptides with Ion Mobility-Mass Spectrometry, J. Proteome Res. 1, 303-306 (2002)].
Contemporary IM and IM-MS is performed by injecting ions into the drift cell slower than the transient rate of ion separation necessary to retain analyte injection/detection time correlation (i.e., at a rate <td−1, where td is the drift time of the ions through the mobility cell). Traditionally this is termed the “pulse-and-wait” approach. However, significant enhancements in signal-to-noise (S/N) and throughput can be realized by adapting multiplex data acquisition methods to IM-MS. Fourier transform (FT), Hadamard transform (HT), and correlation techniques are commonly used in optical and molecular spectroscopy, but their application to mass spectrometry has, until recently, been limited to FT-ion cyclotron resonance-MS [M. Harwit and N. J. A. Sloane, Hadamard Transform Optics, Academic Press, New York, N.Y. (1979); A. G. Marshall, Ed., Fourier, Hadamard, and Hilbert Transforms in Chemistry, Plenum Press, New York, N.Y. (1982); A. G. Marshall and F. R. Verdun, Fourier Transforms in NMR, Optical, and Mass Spectrometry, Elsevier, New York, N.Y. (1990)]. The Fellgett advantage afforded by these techniques can also be realized by injecting ion packets into the IM drift cell or TOFMS drift tube faster than the sequential (i.e., pulse-and-wait) duty cycle. Although both techniques achieve separation based on time dispersion of the analytes, multiplexing of IMS or TOFMS have only been described as distinctly separate experiments.
For example, Hill and coworkers have demonstrated a 1.4-fold increase in IM sensitivity by in-phase frequency sweeping of ion gates (Bradbury-Nielsen design [N. E. Brabury and R. A. Nielsen, Absolute Values of the Electron Mobility in Hydrogen, Phys. Rev. 49, 388-393 (1936)]) at the entrance and exit of the drift cell. The ion mobility arrival time distributions were reconstructed from the frequency-domain interferogram by application of a Fourier transform [F. J. Knorr, R. L. Eatherton, W. F. Siems, and H. H. Hill Jr., Fourier Transform Ion Mobility Spectrometry, Anal. Chem. 57, 402-406 (1985); R. L. Eatherton, W. F. Siems, and H. H. Hill Jr., Fourier Transform Ion Mobility Spectrometry of Barbiturates After Capillary Gas Chromatography, J. High Res. Chrom. Chrom. Commun. 9, 44-48 (1986); R. H. St. Louis, W. F. Siems, and H. H. Hill Jr., Apodization Functions in Fourier Transform Ion Mobility Spectrometry, Anal. Chem. 64, 171-177 (1992); Y.-H. Chen, W. F. Siems, and H. H. Hill Jr., Fourier Transform Electrospray Ion Mobility Spectrometry, Anal. Chim. Acta 334, 75-84 (1996); U.S. Pat. No. 4,633,083 to Knorr, et al.]. Franzen later described fast-FT and fast-HT multiplexing of IM by modulating the ion beam admittance to the drift cell by means of a Bradbury-Nielsen gate [U.S. Pat. No. 5,719,392 to Franzen]. A unique means for performing FT-IMS was also described by Tarver and Siems, whereby a frequency-domain spectrum is obtained by either frequency-sweeping a Bradbury-Nielsen gate and/or frequency-sweeping the detector signal using a fast commutator [U.S. Pat. No. 6,580,068 to Tarver, et al.]. In these different multiplexed IMS experiments it is taught that, by means of their implementation, the duty cycle is only optimally increased to approximately 50%.
Knorr has also described Fourier transform-TOFMS [U.S. Pat. No. 4,707,602 to Knorr]. The FT-TOFMS was equipped with an electron impact ionization source and provided a 25-fold increase in sensitivity over conventional signal-averaging [F. J. Knorr, M. Ajami, and D. A. Chatfield, Fourier Transform Time-of-Flight Mass Spectrometry, Anal. Chem. 58, 690-694 (1986)]. Zare and coworkers have described Hadamard transform-TOFMS to improve the instrumental duty cycle to nearly 50% by using a modulated continuous ESI ion beam with an 8191-order Hadamard matrix [A. Brock, N. Rodriguez, and R. N. Zare, Hadamard Transform Time-of-Flight Mass Spectrometry, Anal. Chem. 70, 3735-3741 (1998); A. Brock, N. Rodriguez, and R. N. Zare, Characterization of a Hadamard Transform Time-of-Flight Mass Spectrometer, Rev. Sci. Inst. 71, 1306-1318 (2000); F. M. Fernandez, J. M. Vadillo, J. R. Kimmel, M. Wetterhall, K. Markides, N. Rodriguez, and R. N. Zare, Hadamard Transform Time-of-Flight Mass Spectrometry: A High-Speed Detector for Capillary-Format Separations, Anal. Chem. 74, 1611-1617 (2002); R. N. Zare, F. M. Fernandez, and J. R. Kimmel, Hadamard Transform Time-of-Flight Mass Spectrometry: More Signal, More of the Time, Angew. Chem. Int. Ed. 42, 30-35 (2003); U.S. Pat. No. 6,300,626 to Brock, et al.]. Zare and colleagues have suggested the possibility of attaining ca. 100% duty cycle by electrostatic steering to modulate and direct the ion beam to different regions of a position sensitive detector [R. N. Zare, F. M. Fernandez, and J. R. Kimmel, Hadamard Transform Time-of-Flight Mass Spectrometry: More Signal, More of the Time, Angew. Chem. Int. Ed. 42, 30-35 (2003).]. Independently, Dowell suggested modulating the ion beam by switching between two sources, or by alternatively modulating a single beam by electrostatic steering and utilizing two detectors [U.S. Pat. No. 5,331,158 to Dowell]. Note that steering modulation in TOFMS dates back to 1948 [A. E. Cameron and D. F. Eggers Jr., Ion “Velocitron,” Rev. Sci. Instrum. 19, 605-607 (1948)], but theoretical and practical implementation was not described until the early 1970s by Bakker [J. M. B. Bakker, A Beam-Modulated Time-of-Flight Mass Spectrometer Part I: Theoretical Considerations, J. Phys. E: Sci. Instrum. 6, 785-789 (1973); J. M. B. Bakker, A Beam-Modulated Time-of-Flight Mass Spectrometer Part II: Experimental Work, J. Phys. E: Sci. Instrum. 7, 364-368 (1974).]. In contrast to FT and HT modes of multiplexing TOFMS, Myerholtz and colleagues have described a technique based on bunching and overlapping ion packets in the field-free drift region and demodulating the resultant signal by using correlation algorithms to improve TOFMS duty cycle to ca. 50% [U.S. Pat. No. 5,396,065 to Myerholtz, et al.].
The present invention differs from the one-dimensional prior art (i.e., IMS or TOFMS) in that significant gains in sensitivity, throughput, and S/N are obtained by two-dimensions of time dispersive analyte ion separation, i.e., by coupling ion mobility-TOFMS and operating both dispersive dimensions in a multiplex data acquisition mode described herein.